In the field of life science, the measurement of an activity of gene transcription which occurs in a cell is generally performed, and used for evaluation of effects of foreign factors which affect the cell, analyses of intracellular signal transduction and expression of an individual protein group. In order to quantitatively determine the gene expression in a wide range and analyze temporal dynamic changes of the gene expression, the measurement of gene transcription activity by reporter technology using luciferase which is a luminescent enzyme has been actively performed. Meanwhile, special substances in vivo have been actively evaluated, quantified and analyzed, and in particular, in immunoassays using antibodies, utilizing recognizability of the antibody, from low molecular biomolecules which are specific hormones and physiologically active substances to high molecular proteins have been quantified. At that time, it is common to detect the antibody by a labeling method utilizing radioactivity or chromogenic property, but in recent years, the labeling method using luciferase has been used. Therefore, it becomes a more important theme to improve and develop the luciferase technology for the purpose of elucidating life phenomena.
As major luminescent proteins currently exploited as the luciferase technology, luciferases derived from firefly, Cypridina, Renilla and copepod, and an aequorin luminescent protein derived from luminescent jellyfish are available. To exploit these luciferases, firefly luciferin, Cypridina luciferin, and coelenterazine (other names: Renilla luciferin, firefly squid preluciferin, oplophorus luciferin) which is a substrate of Renilla luciferase, copepod luciferase and aequorin are available (Table 1). Three luciferins can be directly extracted from luminescent organisms, can also be chemically synthesized and are commercially available.
TABLE 1Relationship of luciferin and luciferaseLuciferaseLuciferinFirefly luciferaseFirefly luciferinBeetle luciferaseCypridina luciferaseCypridina luciferinRenilla luciferaseCoelenterazine (other names:Copepod luciferaseRenilla luciferin, firefly squidOplophorus luciferasepreluciferin, oplophorusLuminescent proteinluciferin)(aequorin)
Coelenterazine which can be synthesized relatively easily has been already used in many products. For example, a dual assay system supplied from Promega is the system which measures two transcription activities with two substrates by inserting a transcription active region A in a firefly luminescent enzyme gene, simultaneously inserting a transcription active region B in a Renilla luciferase gene and introducing two gene constructs in a cell. In this method after lysing the cells, firefly luciferin is added to measure the transcription activity A. The luminescence of firefly luciferase is quenched, and subsequently coelenterazine is added to measure the transcription activity B.
Meanwhile, recently copepod luciferase (Gaussia Luciferase U.S. Pat. No. 6,436,682) extracellularly secreted and supplied from Prolume has been noticed. The system in which this gene is introduced into the cell, and taking advantage of synthesized and secreted luciferase, an expression amount of a gene is measured by adding coelenterazine into medium is suitable for the assay with high throughput.
Since a pKa value of proton of an imidazopyrazinone ring in coelenterazine is around 7.4, the proton is easily dissociated in neutral or weak basic buffer. Thus, coelenterazine reacts with oxygen to release faint light with being oxidized and decomposed. In existing methods, it is necessary to store coelenterazine in an alcohol solution under an acidic condition at pH 4 to 5 in a freezer at −20(C or below (Non-patent literature 1). Meanwhile, the optimal pH of the luciferase group using coelenterazine as the substrate is around the neutral, 7 to 8, and is much different from the pH value which stabilizes coelenterazine. In order to reduce the effect of the alcohol solution under the acidic condition, a small amount of luciferin (1/1000, v/v) relative to the luciferase solution is added (Non-patent literature 1).
Since a volume of a well in a 96-well plate or a 384-well plate used in the assay with high throughput is 0.2 mL or less, it is preferable to add 0.2 (L or less of the coelenterazine acidic alcohol solution (Non-patent literature 1). Since the amount of dispensed liquid in an existing luminometer for the 96-well plate is 25 to 250 (L, it is impossible to add the faint amount of the coelenterazine acidic alcohol solution. In addition, in the case of immunoassay, it is desirable to directly add the coelenterazine solution into the washed well. This way, in any cases, it is necessary to dilute coelenterazine with the buffer at pH 7 to 8.
Meanwhile, since coelenterazine is unstable in a weak basic aqueous solution, when coelenterazine is diluted with the buffer at pH 7 to 8, variation of the luciferin activity between the first sample and the last sample appears in the measurement of samples in a large amount. Thus, the correct measurement can not be performed (FIG. 1). Stability of coelenterazine in the buffer at pH 7 to 8 is an important problem for the assay with high throughput. Upon the measurement of coelenterazine, its self-luminescence which is a background is also a factor to reduce a dynamic range, and a reaction solution to reduce the self-luminescence is required.
Non-Patent literature 1: BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 233, 349-353 (1997)